Supplementary Figures and legends Supplementary Figure S1. Effects of G1069L and G1069W mutations on methyltransferase activity of G9a in vitro (Related to Figure 1).(A) List of human melanomas with a G1069L/W mutation and the source database/study. (B) Co-crystalized structure of G9a and H3K9M mutant peptide obtained from the Protein Data Bank (PDB: 5jin). A part of the co-crystalized structure was visualized by JSmol. G1069 is located immediately adjacent to the highly conserved catalytic site, Y1067, and other histone-binding amino acids.(C) Co-immunoprecipitation of V5-tagged G9a (G9a/V5) and GLP in 293T cells. 24 h after transfection of either G9a wild type (WT) or mutants, nuclear protein fractions were extracted and subsequently used for co-immunoprecipitation using anti-V5 antibody. (D) In vitro methyltransferase assay using GST-fused recombinant G9a and recombinant human H3 protein. Briefly, 2 g of H3 protein was incubated with 2 g of either GST, GST-G9a, GST-G9a G1069L, or GST-G9a G1069W, with or without 1 g of recombinant GLP, in the presence of 2 M S-adenosyl methionine (SAM) for 1h at RT. Subsequently, the mixture was subjected to western blotting using specific anti-methyl H3 antibodies.Coomassie Brilliant Blue (CBB) staining was used as an internal control to ensure similar amounts of the recombinant proteins. (E) Pull-down assay for recombinant human G9a and histone H3 peptides. After elution, histone H3interacting GST-G9a WT and mutant proteins were visualized by immunoblot using anti-GST antibody. (F) Western blots of global H3K9 mono-, di-, and trimethylation levels and H3K27 di-and tri-methylation levels in G9a WT-, G1069L-, and G1069W-overexpressing UACC62 melanoma cells. (G) Western blot of G9a/V5 WT and G1069 mutants in the overexpressing UACC62 cells of F. (H) Effects of G9a WT, G1069L, and G1069W on UACC62 proliferation. Cell numbers were manually counted by trypan blue exclusion assay. **p<0.01, ****p<0.0001 by repeated measures two-way ANOVA of 3 replicates with the Holm-Šidák correction for multiple pairwise comparisons of the four groups at each time point. (I) Effects of G9a/GLP inhibitor UNC0638 on the enhanced proliferation of G9a WT-, G1069L-, and G1069W-expressing UACC62 cells. The cells were cultured in the presence of 1 M UNC0638 for 6 days. Cell numbers were counted manually and normalized to Day 0. *p<0.05, ***p<0.001, ****p<0.0001 by regular two-way ANOVA of 3 replicates with the Holm-Šidák correction for multiple pairwise comparisons. (J) Transformation assay of G9a and oncogenic G1069L/W mutants in NIH3T3 cells. Following infection with the indicated gene, NIH3T3 cells were cultured for 10 days without blasticidin selection. Representative images from one of two independent experiments are shown and average colony numbers +/ SD of 3-4 replicates are indicated below the images. **p<0.01 vs. GFP, § p<0.05 vs. G9a WT by one-way ANOVA with the Holm-Šidák correction for multiple pairwise comparisons.